Nitric oxide (NO), produced by NO synthases (NOS), is an essential signaling molecule with fundamental roles in cardiovascular function, neurotransmission and immune response. Despite significant effort, there are no intact structures of the mammalian NOS holoenzyme and the rearrangements that drive NO synthesis are unknown. Because of this, central questions that are fundamental to the mechanism of NOS remain unanswered, including how large reductase and oxygenase domains converge to transfer electrons and how the Ca2+-calmodulin (CaM) protein interacts to coordinate catalysis. The significant size and flexibility of NOS is a tremendous challenge to structural studies and this has been a roadblock to answering these questions. As a single-particle approach, cryo-electron microscopy (cryo-EM) is a powerful and unexplored method for characterizing this essential enzyme. With the applicant's significant background in cryo-EM, established collaborations with experts in the NOS field, and recently published work elucidating the structure of the neuronal NOS complex, the objective here is: to determine structural and functional mechanisms of the three mammalian NOS isoforms: neuronal (n), endothelial (e) and inducible (i). The central hypothesis is: NO synthesis by NOS requires large domain rearrangements that coordinate with CaM-driven changes in a key FMN subdomain during the electron transfer cycle, and further that the three isoforms have unique structures and conformational states that explain regulatory mechanisms. This hypothesis is strongly supported by our recent work where we determined the first 3D molecular model of the NOS:CaM complex. The Specific Aims for this proposal are to (1) determine the architecture and CaM-dependent structural changes of nNOS by cryo-EM; (2) investigate isoform-specific conformational states of eNOS and iNOS; and (3) characterize structural control elements that define NOS functional states. We have utilized newly developed 2D/3D EM methods and determined a 3D model that identifies a CaM-triggered rotation of the FMN subdomain, which is critical for transferring electrons to the heme. We have determined a preliminary 3D model of iNOS that reveals a CaM-bound conformational state where the reductase and oxygenase domains are aligned in a cross- monomer state. Finally we have developed 2D EM assays to identify the structural basis of key interactions, cis regulatory elements and phosphorylation states that uniquely control the NO synthesis mechanism. The approach is innovative because it integrates the latest methods in cryo-EM imaging and computational analysis with key biochemical techniques to determine the first comprehensive characterization of the NOS holoenzyme complex. The proposed research is significant because it will answer long-standing questions about the mammalian NOS isoforms by determining key structural rearrangements that drive enzyme function. These objectives are a critical foundation to the applicant's long-term goals to understand the structural/functional basis of dynamic macromolecular protein interactions that are critical to cellular signaling and homeostasis.